The worldwide animal feed market, which includes livestock, poultry, aquaculture and pets is 475 million metric tons. In the United States 180 million metric tons are consumed with corn (God mays L.) accounting for about 67% and soybean (Glycine max L.) meal for about 10% of the total. Corn and soybean products are also a major element of foreign trade. These two crops are agronomically well-adapted to many parts of the U.S., and machinery and facilities for harvesting, storing and processing are widely available across the U.S. Because corn, soybean and other crops used for feed are currently sold as commodities, an excellent opportunity exists to upgrade the nutritional quality of the protein and thus add value for the U.S. farmer and enhance foreign trade.
Human food and animal feed derived from many grains are deficient in the sulfur amino acids, methionine and cysteine, which are required in the animal diet. In corn, the sulfur amino acids are the third most limiting amino acids, after lysine and tryptophan, for the dietary requirements of many animals. The use of soybean meal, which is rich in lysine and tryptophan, to supplement corn in animal feed is limited by the low sulfur amino acid content of the legume. Thus, an increase in the sulfur amino acid content of either corn or soybean would improve the nutritional quality of the mixtures and reduce the need for further supplementation through addition of more expensive methionine.
Efforts to improve the sulfur amino acid content of crops through plant breeding have met with limited success on the laboratory scale and no success on the commercial scale. A mutant corn line which had an elevated whole-kernel methionine concentration was isolated from corn cells grown in culture by selecting for growth in the presence of inhibitory concentrations of lysine plus threonine Phillips et al. (1985) Cereal Chem. 62:213-218!. However, agronomically-acceptable cultivars have not yet been derived from this line and commercialized. Soybean cell lines with increased intracellular concentrations of methionine were isolated by selection for growth in the presence of ethionine Madison and Thompson (1988) Plant Cell Reports 7:472-476!, but plants were not regenerated from these lines.
The amino acid content of seeds is determined primarily by the storage proteins which are synthesized during seed development and which serve as a major nutrient reserve following germination. The quantity of protein in seeds varies from about 10% of the dry weight in cereals to 20-40% of the dry weight of legumes. In many seeds the storage proteins account for 50% or more of the total protein. Because of their abundance plant seed storage proteins were among the first proteins to be isolated. Only recently, however, have the amino acid sequences of some of these proteins been determined with the use of molecular genetic techniques. These techniques have also provided information about the genetic signals that control the seed-specific expression and the intracellular targeting of these proteins.
A number of sulfur-rich plant seed storage proteins have been identified and their corresponding genes have been isolated. A gene in corn for a 15 kD zein protein containing 11% methionine and 5% cysteine Pedersen et al. (1986) J. Biol. Chem. 261:6279-6284! and a gene for a 10 kD zein protein containing 23% methionine and 3% cysteine have been isolated Kirihara et al. (1988) Mol. Gen. Genet. 21:477-484; Kirihara et al. (1988) Gene 71:359-370!. Two genes from pea for seed albumins containing 8% and 16% cysteine have been isolated Higgins et al. (1986) J. Biol. Chem. 261:11124-11130!. A gene from Brazil nut for a seed 2S albumin containing 18% methionine and 8% cysteine has been isolated Altenbach et al. (1987) Plant Mol. Biol. 8:239-250!. Finally, from rice a gene coding for a 10 kD seed prolamin containing 19% methionine and 10% cysteine has been isolated Masumura et al. (1989) Plant Mol. Biol. 12:123-130!.
There have been many reports on the expression of seed storage protein genes in transgenic plants. The high-sulfur 2S albumin from Brazil nut has been expressed in the seeds of transformed tobacco under the control of the regulatory sequences from a bean phaseolin storage protein gene. The protein was efficiently processed from a 17 kD precursor to the 9 kD and 3 kD subunits of the mature native protein. The accumulation of the methionine-rich protein in the tobacco seeds resulted in an up to 30% increase in the level of methionine in the seeds Altenbach et al. (1989) Plant Mol. Biol. 13:513-522!. Chimeric genes linking the coding regions of corn seed storage protein genes for 19 and 23 kD zeins to the Cauliflower Mosiac virus 35S promoter were expressed at very low levels in seeds, as well as roots and leaves, of transformed tobacco Schernthaner et al. (1988) EMBO J. 7:1249-1255!. Replacement of the moncot regulatory regions (promoter and transcription terminator) with dicot seed-specific regulatory regions resulted in low level seed-specific expression of a 19 kD zein in transformed petunia Williamson et al. (1988) Plant Physiol. 88:1002-1007! and tobacco Ohtani et al. (1991) Plant Mol. Biol. 16:117-128!. In another case, high-level seed-specific expression of the 15 kD sulfur-rich zein was found in transformed tobacco, and the signal sequence of the monocot precursor was also correctly processed Hoffman et al. (1987) EMBO J. 6:3213-3221!.
In order to increase the sulfur amino acid content of seeds it is essential to isolate a gene(s) coding for seed storage proteins that are rich in the sulfur-containing amino acids methionine and cysteine. Methionine is preferable to cysteine because methionine can be converted to cysteine, but cysteine cannot be converted to methionine by most animals. It is desirable that the storage protein be compatible with those of the target crop plant. Furthermore, it is desirable that the protein come from a source that is generally regarded as safe for animal feed.